1. Field of the Invention:
This invention relates to and has among its objects the provision of novel methods for the preparation of highly-purified human Factor VIII:C and of a novel procoagulant material having substantially less carbohydrate than native human Factor VIII:C.
2. Description of the Prior Art:
It is known that the clotting of human blood is a complicated process, involving a series of reactions mediated by 13 different factors. It also is well known that a cause of hemophilia is the inability of the afflicted individual to synthesize one of these factors, known variously as antihemophilic factor, AHF, AHG, Factor VIII or Factor VIII C, in amounts sufficient to support adequate clotting. Dried preparations of AHF concentrate are sold commercially for administration to hemophiliacs for treatment of bleeding or in advance of surgery. The AHF concentrate is obtained from plasma from human donors, through the use of known techniques.
The usual commercial AHF preparation is not pure Factor VIII:C. Rather, it is an AHF-enriched fraction obtained from plasma. It is desirable that the AHF concentrate be as pure as possible, but further improvements in purity through modification of the procedure for isolating AHF from plasma have not been practically feasible due to the difficulty of separating plasma components. AHF is quite difficult to separate and purify because of its low content in the plasma and the instability of its activity. The known AHF concentrates are prepared from fraction I separated from plasma by means of Cohn's ethanol fractionation method or from the cryoprecipitate obtained by freezing a plasma and then thawing it at a low temperature. However, they are all crude products of low purity and contain a large quantity of fibrinogen.
As mentioned above, the AHF concentrates obtained by the prior art processes discussed above are of relatively low specific activity, namely about one unit or less of AHF activity per milligram (mg) of protein, one of the undesirable impurities being denatured AHF. U.S. Pat. Nos. 4,289,691 and 4,302,445 disclose processes for preparing AHF preparations having a specific activity of AHF activity of 1-3 units per mg of protein, and U.S. Pat. No. 4,294,826 describes a method for preparing human AHF having a specific activity of about 1-10 units of AHF activity per mg.
AHF, or Factor VIII, is now known, however, to exist as a complex of several different protein subunits with several different functions. The exact nature of the complex (i.e. whether the subunits are separate molecules or parts of one molecule and which functions are associated with which subunits) is not yet fully understood. However, it is now known that procoagulant activity, or AHF, or antihemophiliac activity is associated with one subunit, termed Factor VIII:C. Von Willebrand activity, measured as platelet aggregation activity, is associated with a subunit termed Factor VIII:vWF or Factor VIII R:Ag. Another functional subunit is termed Factor VIII C:Ag since this subunit contains the F VIII antigen.
Several investigators have tried to separate F. VIII:C from other active Factor VIII components, as disclosed in Fulcher et al, P.N.A.S., 79:1648-152 and the related patent, U.S. Pat. No. 4,361,509, cited in the copending application. Carbohydrate side chains have been studies in multimeric Factor VII:C/vWF to determine their role in platelet aggregation. In addition, alteration of these side chains is known to affect plasma clearance. These side chains are thought to terminate in sialic acid residues linked to galactosyl residues. In mammals, a specific hepatic receptor recognizes terminal galactosyl residues on glycoproteins which have been treated to remove sialic acid, so that these asialoglycoproteins are cleared rapidly by the liver.
Sodetz et al, J. Biol. Chem., 252:5538-46, (1977) disclose the neuramidase treatment of human F. VIII:C/vWF purified from human AHF concentrates. They found that upon removal of sialic acid, vW (platelet aggregating) activity is markedly reduced, while procoagulant activity remains constant. The circulating half-life of the treated protein in rabbits goes from about 240 min. to about 5 min.
Sodetz et al, J. Biol. Chem., 253:7202-7206, (1978) report treatment of F. VIII:vWF with neuramidase as in the previous experiment, followed by incubation with galactosidase to remove 62% of the galactose. Procoagulant activity was not decreased, but VW activity dropped markedly.
From these studies it was concluded that vWF activity is dependent on terminal sialic acid residues, the penultimate galactose residues, and protein structure.
Gralnick et al, P.N.A.S., 80:2771-2774, (1983) treated F. VIII/vWF with neuramidase beta galactosidase, and galactose oxidase. They reported, consistently with Sodetz et al, that this treatment reduced vWF activity. It was suggested that the next-to-terminal galactose is responsible for maintaining the largest multimers of the factor VIII/vWF factor protein. Treatment of intact protein with these enzymes did not produce a lowering of vWF or procoagulant activity, but treatment of the asialo factor VIII/vWF protein with beta galactosidase resulted in a time-dependent decrease of vW factor activity. This was correlated with loss of the largest multimeric subunits and vW activity.